Abnormality in the Optic Nerve of Albino Mutant Quails

Abnormality in the Optic Nerve of Albino Mutant Quails Koichi Takarsuji* and Akira Nokamurof Comparative studies were made between the optic nerves of albino and normal quails. The ipsilateral and contralateral retinofugal projections in the albino quail were similar to those in the normal quail. The abnormality of uncrossed projections in the visual pathways, which is found in albinos of many different mammalian species, was not present in the albino quail. The total cross-sectional area of the optic nerve was approximately 25% smaller in the albino quail than in the normal quail, whereas the fiber density was about 17% higher in the former. The total number of optic nerve fibers in normal and albino quails was therefore the same. Numerous unmyelinated fibers existed in normal and albino quail optic nerves, the ratio of myelinated to unmyelinated fibers being approximately 4:6. Axon diameters of myelinated and unmyelinated fibers in the optic nerves were smaller for the albino quail than for the normal quail. The number of the myelin rings in the albino quail slightly decreased compared with those in the normal quail. Invest Ophthalmol Vis Sci 28:384-390, 1987 Various functional and morphological abnormalities in the visual pathways of several albino mammalian species have been reported. 1 " 7 Morphologically, reduced ipsilateral retinal fibers and an abnormally laminated dorsal lateral geniculate nucleus have been observed in albino species. The abnormalities in the visual pathways are related to the lack of pigment in the retinal pigment epithelium. 89 In contrast to the abnormality in albino mammals, no reduction of ipsilateral retinofugal projections is found in albino amphibians. 10 The mutant quail described here is an imperfect albino (gene symbol al) mutation with a sex-linked recessive gene." 12 The mutant quail has white feathers, except for its back, and a red eye instead of brown. There is little pigment in the pigment epithelium, choroid and pecten oculi, whereas some pigment granules are observed in the ora serrata, ciliary processes, and iris. Albino mutant quails that exhibit interesting ocular diseases, such as eye enlargement, retinal ganglion cell degeneration, and cupping of the optic disc during the aging process, were recently found. l3 These pathological changes in the eyes were related to reduced corneal curvature and lens enlargement. 1415 However, no retinal ganglion cell degeneration is observed in albino quail eyes until about 3 months after hatching. In this study, we compared ipsilateral retinofugal projections, From the Department of Anatomy,* Osaka University Medical School, Osaka and Shizuoka Women's College,f Hamamatsu, Japan. Submitted for publication: August 28, 1985. Reprint requests: Koichi Takatsuji, DMSc, Osaka Prefectural College of Nursing, 2-1-41, Tezukayamahigashi, Sumiyoshiku, Osaka 558,Japan. axon diameters, and myelin rings in the optic nerve of normal and unaffected albino quails. Materials and Methods Normal and albino quails (Coturnix coturnix japonica) were kept under continuous 20-W incandescent bulbs for 4 wk following hatching. They were then exposed to a diurnal schedule, usually 16 hr of light per day, and became mature at 2 months after hatching. The quails were fed a quail diet and water ad libitum, and used in the experiments at 2-3 months after hatching. Eight albino and five normal quails were used for the study of the retinal projections by the anterograde horseradish peroxidase (HRP) method. Under anesthesia with sodium pentobarbital (2.5 mg/100 g, intraperitoneally), the cornea, lens, vitrous body, and pecten oculi were removed from one eye. A total volume of 3-5 n\ of a 30-50% solution of HRP (Sigma Chemical; St. Louis, MO, Type VI) was injected into the optic nerve head. Injections were made with a glass micropipette connected to a syringe by a plastic tube. After the injection, the opening in the eyeball was sealed with acrylic resin. Two days after the injection, the quails were anesthetized with sodium pentobarbital and intracardially perfused with 0.1 M phosphate buffer (ph 7.4) followed by a fixation solution consisting of 1% paraformaldehyde and 2% glutaraldehyde in 0.1 M phosphate buffer (ph 7.4). Brains were removed, postfixed in the same fresh fixative for 12 hr, and rinsed in 0.1 M phosphate buffer (ph 7.4) containing 30% sucrose for 24 hr. Serial frontal sections were cut at 50 jim with a cryostat and reacted with diaminobenzidine 384

No. 2 ABNORMALITY IN THE ALBINO QUAIL OPTIC NERVE / Takarsuji and Nakamura 385 following the procedure of Adams. 16 After the reaction, the sections were mounted on slides with 0.75% gelatin in water, and dried for 24 hr at room temperature. Sections were counterstained with 1% neutral red for histological identification. Seven albino and seven normal quails, each weighing about 100 g, were used for the study of optic nerve fibers. The quails were anesthetized with sodium pentobarbital and then perfused with 2% paraformaldehyde-2% glutaraldehyde in 0.1 M phosphate buffer (ph 7.4). The optic nerve was taken out together with the eyeball. A small incision was made on the top of the eyeball to ensure correct orientation. The tissue specimen was rinsed with 0.1 M phosphate buffer (ph 7.4) for 30 min, postfixed with 2% osmium tetroxide in 0.1 M phosphate buffer (ph 7.4), dehydrated in alcohol, and embedded in Epon 812. Semithin sections were taken from the middle part of the optic nerve and stained with a toluidine blue solution for measurement of a cross-sectional area. Ultrathin sections were stained with 0.1% lead citrate and examined with a Hitachi HU-11B electron microscope (Hitachi, Japan). For measurement of axon diameters, a cross-sectioned optic nerve was divided into 12 regions, and electron micrographs were taken from each region. Electron micrographs were taken at an original magnification of 7,000 with photographic enlargement of 3 (final magnification: 21,000). Axon areas were measured using an image analyzer (Mutoh Company, Japan). Axon diameters were calculated using the following equation (L = diameter; S = cross-sectional area of axon): L = 2 The area of the cross-sectioned optic nerve was also measured in freshly enucleated eyes of some quails for estimation of areal shrinkage due to histological procedures. Some electron micrographs were obtained with an original magnification of 20,000 and the same photographic enlargement (final magnification: 60,000) to count the myelin rings. All measured values obtained were analyzed statistically by a computer (Canon BX-1, Japan). Animal care and treatment in this investigation were in compliance with the ARVO Resolution on the Use of Animals in Research. Retinal Projections Results The nomenclature adopted for the relevant subdivision of the diencephalon and mesencephalon related to the optic pathways followed that used for pigeons. 17 In normal and albino quails, the majority of anterogradely HRP-labeled retinal fibers decussated in the SGF Fig. 1. A-C: a series of tracings of selected sections taken from the albino quail brain. The retinal projections arising from the HRPinjected eye are indicated in a semischematic fashion. Fibers anterogradely labeled are shown by dashed lines. Labeling in terminal areas is indicated by dots. The left side of each section is ipsilateral to the injected eye. DLL, nucleus dorsolateralis anterior thalami pars lateralis; EM, nucleus ectomamillaris; GLv, nucleus geniculatus lateralis pars ventralis; LA, nucleus lateralis anterior thalami; LH, nucleus lateralis hypothalami; LM, nucleus lentiformis mesencephali pars magnocellularis; OT, optic tectum; Rt, nucleus rotundus; SGF, stratum griseum et fibrosum superficiale; TO, tractus opticus; TrEM, tractus nuclei ectomamillaris; Bar, 1 mm. optic chiasm and projected to the contralateral diencephalic and mesencephalic visual relay nuclei, ie, nucleus anterior lateralis thalami, nucleus geniculatus lateralis pars ventralis, nucleus dorsolateralis anterior thalami pars lateralis, nucleus lentiformis mesencephali pars magnocellularis, nucleus pretectalis diffusus, nucleus ectomamillaris, and optic tectum (Fig. 1). Each of the many diencephalic and mesencephalic structures had been identified by Cowan et al 18 and Reperant 19 as being innervated by retinal afferents. No significant differences were observed in the contralateral retinal projections between normal and albino quails. A small population of retinal fibers projected to the ipsilateral diencephalic and mesencephalic visual relay nuclei in normal quails. Most of the ipsilateral retinal fibers traveled caudolaterally in the anterior optic fascicle of Reperant or tractus opticus, and projected to

386 INVESTIGATIVE OPHTHALMOLOGY G VISUAL SCIENCE / Februory 1987 Vol. 2 8 Table 1. Comparison in a cross-sectional area of the optic nerve, axon diameters of myelinated fibers and unmyelinated fibers, and the number of myelin rings of the optic nerve fibers between normal and albino quails* Fig. 2. Anterogradely HRP-labeled ipsilateral fibers (arrowheads) in the tract opticus (TO) or anterior optic fascicle of the albino quail; LH, nucleus lateralis hypothalami; Bar, 100 nm. the nucleus anterior lateralis thalami, nucleus geniculatus lateralis pars ventralis and rostral part of the nucleus dorsolateralis anterior thalami pars lateralis. Some of the HRP-labeled fibers on the injection side entered the tractus nuclei ectomamillaris. These fibers reached the nucleus ectomamillaris. The ipsilateral retinal projections in normal quails were in agreement with those described previously.20 In albino quails, several retinal fibers also projected ipsilaterally (Fig. 1). Most of the HRP-labeled retinal fibers in the ipsilateral anterior optic fascicle or tractus opticus ran caudolaterally and projected to the nucleus anterior lateralis thalami, rostral part of the nucleus dorsolateralis anterior thalami pars lateralis, and nucleus geniculatus lateralis pars ventralis (Fig. 2). Some fibers left the medial aspect of this fascicle to entered the nucleus lateralis hypothalami. The fibers may be terminating in the hypothalamus. Some of the HRPlabeled retinal fibers traveled ipsilaterally in the tractus Fig. 3. Anterogradely HRP-labeled ipsilateral fibers (arrowheads) in the nucleus ectomamillaris (EM) of the albino quail. A high power photograph taken in the area of the broken line is shown in the upper left; SO, stratum opticum of the optic tectum; Bar, 100 fim. Cross-sectional area (mm2) Axon diameter of myelinated fibers (^m) Axon diameter of unmyelinated fibers (fim) Number of myelin rings Normal Albnino P (nest) 1.35 ±0.05 1.07+0.10 <0.05 0.65 ±0.23 0.48 ±0.18 <0.001 0.26 ±0.08 0.20 ±0.05 <0.001 7.31 ±2.42 6.21 ±2.32 <0.05 * Values are expressed as mean ± SD. nuclei ectomamillaris, projected to the nucleus ectomamillaris, and ramified within the nucleus (Fig. 3). No HRP-labeled retinal fibers were observed in the ipsilateral optic tectum of normal and albino quails. In summary, ipsilateral retinal projections in albino quails were similar to those in normal quails. Optic Nerve Fibers The optic nerve of the albino quail was slender to the naked eye compared with that of the normal quail. In the optic nerve, bundles of fibers were separated by connective tissue, glial nuclei were prominent, and small blood vessels were widely distributed. A total cross-sectional area of the optic nerve was measured in eight specimens of normal quails and in eight specimens of albino quails. Table 1 shows the cross-sectional area in the optic nerve. The average area was 1.35 ± 0.05 mm 2 in the normal quail and 1.07 ± 0.10 mm 2 in the albino quail. In freshly enucleated eyes, the cross-sectional area of the optic nerve was 1.40 ±0.15 mm 2 in the normal quail and 1.10 ± 0.13 mm 2 in the albino quail. Shrinkage was small in paraformaldehyde- and glutaraldehyde-fixed, osmium tetroxidepostfixed, and Epon-embedded specimens. The total cross-sectional area of the optic nerve in the albino quail was, on the average, about 25% smaller than that in the normal quail. Each three cross-section obtained from normal and albino quail optic nerves was used for the systematic measurement of axon diameter, which was done using 12 electron microscopic photographs. The electron microscopic photographs covered a total area of 988 fim2 in each optic nerve. A total of 8,334 axons in the normal quail and 10,005 axons in the albino quail were counted from the photographs. Viewed in the electron microscope, the quail optic nerve exhibited intermingling of myelinated and unmyelinated fibers (Figs. 4 A

No. 2 ABNORMALITY IN THE ALDINO QUAIL OPTIC NEtWE / Takarsuji and Nakomuro 387 Fig. 4. Electron micrographs of cross-sections in normal (A) and albino (B) quail optic nerves. Myelinated and unmyelinated fibers are intermingled in normal and albino quails; Bar, 1 ^m. and 4B). In normal and albino quails, many unmyelinated fibers existed in the optic nerve, the ratio of myelinated to unmyelinated fibers being approximately 4:6. Figure 5 shows the histograms of the axon diameters of myelinated fibers in normal and albino quails. The axon diameters ranged from 0.17-1.68 ^m. The distributions appeared to be primarily unimodal with a mean fiber diameter of 0.65 fxm in the normal quail and 0.48 ^.m in the albino quail. The histogram for the albinos showed a sharper peak than that for the normal quail. Figure 6 shows the histograms of the axon diameters of unmyelinated fibers in normal and albino quails. The axoplasm contained many neurotubules and neurofilaments, which were useful for distinguishing unmyelinated axons from glial processes. The histograms of unmyelinated fibers had a single mode at 0.2-0.3

388 INVESTIGATIVE OPHTHALMOLOGY G VISUAL SCIENCE / Februory 1987 Vol. 28 20 NORMAL N=2891 X±S.D=0.65*0.23 is- le- ALBINO N-3312 X±S.D=0.4810.18 10- Fig. 7. Histograms showing the number of myelin rings per nerve fiber in a sample of 200 myelinated fibers in normal (thick line) and albino (fine line and dots) quails. 10 15 05 1.0 1.5 Fig. 5. Histograms of myelinated axon diameter of the optic nerve in normal (thick line) and albino (fine line and dots) quails. nm in the normal quail, but it showed a single mode at 0.15-0.25 nm in the albino quail. The mean axon diameter of unmyelinated fibers in the albino quail was also slightly smaller than that in the normal quail, being 0.20 urn and 0.26 nm, respectively. A significant difference was found in the axon diameter of the myelinated and unmyelinated fibers between normal and albino quails (Table 1). Many myelinated fibers had five to nine myelin rings. The degree of myelination expressed by the myelin 40 30 20 10 0 3 0 6 NORMAL N=4598 X±S.D=0.2610.08 ALBINO N=5632 X±S.D=0.2010.05 Fig. 6. Histograms of unmyelinated axon diameter of the optic nerve in normal (thick line) and albino (fine line and dots) quails. rings is shown in Figure 7. The number of the myelin rings of optic nerve fibers in the albino quail was slightly lower than that in the normal quail, and the mean value (0.049 nm) of the myelin sheath thickness in the albino quail was slightly smaller than that (0.059 (im) in the normal quail. The total number of optic nerve fibers was estimated from a multiplication of the number per unit area (in the count area) by the entire cross-sectional area of the optic nerve. The total fiber count ranged from 2,360,000 to 2,720,000 in the normal quail and from 2,180,000 to 2,750,000 in the albino quail. Discussion Albinism frequently results in some defects in the eyes and in the visual systems of vertebrates. 1 " 7-21 The albino quail described here develops several diseases of the eye during the aging process. 13 " 15 Avian and amphibian species are important experimental animals to study the developmental mechanisms, gene effects, and environmental effects in eye abnormalities, since early eye transplants are possible in these species. Although the abnormality of the ipsilateral retinal projections has been reported in many albino mammalian species, the abnormality in retinofugal projections does not occur in albino axolotls. 10 Similarly, in the present study, no significant differences were found in the ipsilateral retinal projections between normal and albino quails. The ipsilateral retinofugal component is small in adult avian and amphibian species, 10 ' 2022 which may be related to the absence of a notable defect in ipsilateral pathways. The total number of fibers in the optic nerve was reported in pigeons to be 2,380,000, (Binggeli and Paule), 23 or 2,300,000 (Duff and Scott); 24 2,430,000-

No. 2 ABNORMALITY IN THE ALBINO QUAIL OPTIC NERVE / Tokorsuji and Nakamura 389 2,630,000 in chickens (Rager and Rager); 25 and 1,500,000 in ducks (O'Flaherty). 26 The total number of fibers in the quail optic nerve is similar to that in avian species studied except for ducks. The optic nerve contains 2,360,000-2,720,000 fibers in the normal quail and 2,180,000-2,750,000 fibers in the albino quail. Most of these are retino-fugal, but the avian optic nerve also contains centrifugal fibers, the cell bodies of which are located in the isthmo-optic nucleus. A total of 8,000-12,000 axons coursing centrifugally in the pigeon optic nerve has been estimated on the basis of cell counts of the isthmo-optic nucleus. 27 This comprises less than one-half of 1% of our total count. The axon diameters in the quail optic nerve were found to be smaller than those in the pigeon or duck optic nerve. This discrepancy might be due to the difference in species. The optic nerve fibers in pigeons and ducks are mostly myelinated. 24 ' 25 However, in the present study, numerous unmyelinated fibers existed in normal and albino quail optic nerves behind the lamina cribrosa. The ratio of myelinated and unmyelinated fibers was 4:6 in normal and albino quails. The differences in the axonal myelination in the optic nerve of quails, and pigeons or ducks were notable. In further comparisons, most axons in anurans optic nerve are unmyelinated, 28 whereas most axons in mammalian optic nerve are myelinated. 29 " 31 The optic nerve in the turtle contains a mixture of unmyelinated and myelinated axons. 32 The intermingled myelinated and unmyelinated fibers in the quail optic nerve may thus represent primitive features in the optic nerve of avian species. The optic nerve of the albino quail was about 25% smaller in cross-sectional area than that of the normal quail. Since the total number of optic nerve fibers in normal and albino quails was the same, and the features of the ganglion cell layer in albino quail retina were similar to those in the normal quail retina, 13 it appears that there is not a selective loss of large or a preservation of small ganglion cells in the albino quail. Therefore, the smaller cross-sectional area in the albino quail optic nerve could be explained by a smaller axon diameter as well as by a thinner myelin sheath. Decreased axon diameter and myelin sheath thickness were reported in the albino rat optic nerve. 31 The albino mutation may therefore influence the development of the optic nerve axons and their myelination. Key words: albino quail, optic nerve, axon diameter, myelin rings, ipsilateral fibers, visual system Acknowledgment We thank Dr. Yutaka Fukuda, Osaka University Medical School, for his valuable advice. References 1. Birch D and Jacobs GH: Spatial contrast sensitivity in albino and pigmented rats. Vision Res 19:933, 1979. 2. Creel DJ, Dustman RE, and Beck EC: Differences in visually evoked responses in albino versus hooded rats. Exp Neurol 29: 298, 1970. 3. Guillery RW: An abnormal retinogeniculate projection in Siamese cats. Brain Res 14:739, 1969. 4. Guillery RW, Scott GL, Cattanach BM, and Deol MS: Genetic mechanisms determining the central visual pathways of mice. Science 179:1014, 1973. 5. Guillery RW, Okoro AN, and Witkop CJ Jr: Abnormal visual pathways in the brain of a human albino. Brain Res 96:373, 1975. 6. Guillery RW, Oberdorfer MD, and Murphy EH: Abnormal retino-geniculate and geniculo-cortical pathways in several genetically distinct color phases of the mink (Muslela vison) J Comp Neurol 185:623, 1979. 7. Lund RD: Uncrossed visual pathways of hooded and albino rats. Science 149:1506, 1965. 8. Silver J and Sapiro J: Axonal guidance during development of the optic nerve: the role of pigmented epithelia and other extrinsic factors. J Comp Neurol 202:521, 1981. 9. Strongin AC and Guillery RW: The distribution of melanin in the developing optic cup and stalk and its relation to cellular degeneration. J Neurosci 1:1193, 1981. 10. Guillery RW and Updyke BV: Retinofugal pathways in normal and albino axolotls. Brain Res 109:235, 1976. 11. Lauber JK: Sex-linked albinism in the Japanese quail. Science 146:948, 1964. 12. Nakamura A and Kaneko T: Preliminary note on sex-linked albinism in the Japanese quail (Coturnix coturnix japonica). Shizuoka Women's College Bulletin 20:69, 1974. 13. Takatsuji K, Ito H, Watanabe M, Ikushima M, and Nakamura A: Histopathological changes of the retina and optic nerve in the albino mutant quail (Commix coturnix japonica). J Comp Pathol 94:387, 1984. 14. Takatsuji K, Iizuka S, Nakatani H, and Nakamura A: Morphology of the cataract in albino mutant quails (Coturnix coturnix japonica). Exp Eye Res 40:567, 1985. 15. Takatsuji K, Sato Y, Iizuka S, Nakatani H, and Nakamura A: Animal model of closed angle glaucoma in albino mutant quails. Invest Ophthalmol Vis Sci 27:396, 1986. 16. Adams JC: Heavy metal intensification of DAB-based HRP reaction production. J Histochem Cytochem 29:775, 1981. 17. Karten HJ and Hodos W: A Stereotaxic Atlas of the Brain of the Pigeon (Columba livid). Baltimore, The Johns Hopkins Press, 1967. 18. Cowan WM, Adamson L, and Powell TPS: An experimental study of the avian visual system. J Anat 95:545, 1961. 19. Reperant J: Nouvelles donnees sur les projections visuelles chez le pigeon (Columba livia). J Hirnforsch 14:151, 1973. 20. Takatsuji K, Ito H, and Masai H: Ipsilateral retinal projections in Japanese quail, Coturnix coturnix japonica. Brain Res Bull 10:53, 1983. 21. Pearson ES: Congenital eye anomalies in albino mice. Nature 114:443, 1924. 22. O'Leary DDM, Gerfen CR, and Cowan WM: The development and restriction of the ipsilateral retinofugal projection in the chick. Dev Brain Res 10:93, 1983. 23. Binggeli RL and Paule WJ: The pigeon retina: quantitative aspects of the optic nerve and ganglion cell layer. J Comp Neurol 137: 1, 1969.

390 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / February 1987 Vol. 28 24. DuffTA and Scott G: Electron microscopic evidence of a ventronasal to dorsotemporal variation in fiber size in pigeon optic nerve. J Comp Neurol 183:679, 1979. 25. Rager G and Rager U: Systems-matching by degeneration: I. A quantitative electron microscopic study of the generation and degeneration of retinal ganglion cells in the chicken. Exp Brain Res 33:65, 1978. 26. O'Flaherty JJ: The optic nerve of the mallard duck: fiber-diameter frequency distribution and physiological properties. J Comp Neurol 143:17, 1971. 27. Cowan WM and Powell TPS: Centrifugal fibres in the avian visual system. Proc R Soc Lond 158:232, 1963. 28. Maturana HR: Number of fibers in the optic nerve and the number of ganglion cells in the retina of anurans. Nature 183:1406, 1959. 29. Hughes A and Wassle H: The cat optic nerve: fibre total count and diameter spectrum. J Comp Neurol 169:171, 1976. 30. Potts AM, Hodges D, Shelman CB, Fritz KJ, Levy NS, and Mangnall Y: Morphology of the primate optic nerve. I. Method and total fiber count. Invest Ophthalmol 11:980, 1972. 31. Sugimoto T, Fukuda Y, and Wakakuwa K: Quantitative analysis of a cross-sectional area of the optic nerve: a comparison between albino and pigmented rats. Exp Brain Res 54:266, 1984. 32. Woodbury PB and Ulinski PS: Conduction velocity, size and the organization of retinal ganglion cell axons in the turtle, Pseudemys scripta. Am Zool 24:48, 1984.