Chemical and ultrastructural changes in tapetum of beagles with a hereditary abnormality

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1 Chemical and ultrastructural changes in tapetum of beagles with a hereditary abnormality Guang Y. Wen, John A. Sturman, Henryk M. Wisniewski, A. MacDonald, and Wendell H. Niemann We have defined the defect in the tapetum of beagles with an inherited tapetal abnormality to be the absence of both zinc and cysteine from the tapetal rods. This was demonstrated by direct measurement of zinc and cysteine and by histochemical localization of zinc. The latter method also indicated an interesting substructure in tapetal rods from normal beagles, in which the zinc is apparently distributed in two concentric rings. The chemical changes involved in different methods of fixing this unique tissue have been examined and correlated with the ultrastructure. (INVEST OPHTHALMOL VIS SCI 23: , 1982.) Key words: tapetum, beagle, cysteine, zinc, taurine, fixation The tapetum lucidum is a reflecting layer situated in the eyes of a number of vertebrates. It is located behind the photoreceptors and gives them a second opportunity to absorb light not initially absorbed (see ref. 1 for review). Our interest in the physiologic role of taurine and the recent discovery that dietary taurine deprivation caused disorganization and degeneration of the tapetum lucidum of the cat 2 led to the present investigation of a naturally occurring tapetal abnormality in the beagle, first described by Bellhorn et al. 3 From the Department of Pathological Neurobiology, N.Y.S. Institute for Basic Research in Developmental Disabilities, Staten Island, N. Y., and Department of Experimental Pathology and Toxicology, Hoffmann- La Roche, Inc., Nutley, N. J. This research was supported by the Office of Mental Retardation and Developmental Disabilities of the State of New York, and by NIH Public Health Service Grant HD Submitted for publication Dec. 15, Reprint requests: Dr. G. Y. Wen, Department of Pathological Neurobiology, N.Y.S. Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Rd., Staten Island, N. Y This condition appears to be inherited as an autosomal recessive trait, a conclusion that is supported by the additional information presented here. Beagles with this condition do not have an ophthalmoscopically visible tapetum but rather have a uniform brownish-red fundus reflex. The original study clearly demonstrated that such dogs did have a histologically verifiable tapetum lucidum, and an electron micrograph of a 13-week-old female showed that the tapetal cells were normal in outline although they lacked the regular array of tapetal rods that are normally present in dogs. Instead, the abnormal tapetal cells appear to contain various-sized inclusions surrounded by a membrane (possibly "empty" tapetal rods) and some electrondense inclusion bodies that appear to be spherical or ellipsoidal in shape. These last structures resemble some features described in the degenerating tapetal cells of the taurine-depleted cat. 2 This article reports a more detailed examination of the chemistry of the tapetum in the beagle with an inherited tapetal abnormality and correlates it with the ultrastructural ob /82/ / Assoc. for Res. in Vis. and Ophthal., Inc. 733

2 734 Wen et al. Invest. Ophthalmol. Vis. Sri, December 1982 Fig. 1. Electron micrographs of cross sections of center of tapetum from normal beagle (a and c) and beagle with inherited tapetal abnormality (b and d). These tissues, fixed by immersion in osmium tetroxide, show the tapetal cells of normal beagles to contain regular arrays of electron-dense profiles surrounded by a membrane. The abnormally shaped tapetal cells from beagles with the inherited abnormality are not obviously different with this fixation from that illustrated in Fig. 2. (a and b, bars = 1 fxm; c and d, bars = 0.1 /Jtm.) servations. In addition, we have examined the differences observed by electron microscopy resulting from different methods of fixation and staining and have correlated them with the chemistry involved in the various reactions. Materials and methods Animals. The beagles used ranged in age from 10 months to 2 years. During this study, we examined nine beagles with abnormal tapeta (presumably homozygous recessive), 12 homozygousdominant normal beagles (obtained from breeding stock in which the tapetal abnormality had never been observed), and four beagles known to be heterozygotes for this condition (bred from females homozygous for this tapetal abnormality). Tissue preparation for electron microscopy (EM). The appearance of the dog tapetum in the electron micrographs varied greatly with the fixation methods employed. 4 " 6 Therefore the follow-

3 Volume 23 Number 6 Chemical changes in tapetum of beagles 735 Fig. 2. Electron micrographs of cross sections of center of tapetum from normal beagle (a and c) and beagle with inherited tapetum abnormality (b and d). These tissues, fixed with glutaraldehyde followed by treatment with osmium tetroxide, show the tapetal cells of normal beagles to contain regular arrays of tubular profiles. The abnormally shaped tapetal cells from beagles with the inherited abnormality are filled with various-sized membrane-bound vacuoles, and the regular arrays are conspicuously absent. The irregularly shaped tapetal cells from the abnormal beagles are separated by quantities of organized material resembling collagen, (a and b, bars = 1 fxm; c and d, bars = 0.1 fxm.) ing fixation methods were used. All dogs were anesthetized with pentobarbital (Nembutal). Perfusion fixation with glutaraldehyde. The animals were killed by intracardiac perfusion (1% paraformaldehyde and 2% glutaraldehyde in 0.1 M S0rensen's phosphate buffer, ph 7.4, for 2 min, followed by 4% glutaraldehyde in the same buffer for 15 min). Eyes were removed, hemisected, and stored in 4% glutaraldehyde for 2 to 24 hr before postfixation with 1% osmium tetroxide in the same buffer. They were finally processed lor EM as described previously. 7 Direct immersion fixation with glutaraldehyde or osmium tetroxide. In some cases, one eye of a dog was surgically removed and hemisected before perfusion. After the vitreous was removed, the tapetum was cut and divided into two pieces. One piece was directly immersed in 4% glutaral-

4 'COO \ Zn Zinc Cysteinate 736 Wen et al. Invest. Ophthalmol. Vis. Sci. December 1982 HOOC NH 2 S 0.9' jjmoles Cysteine alone 0.8 H,C X " Zinc-Cysteine Complex CH NrT N 2 C00H HS CH, CH 0.25 pmoles Cysteine 0.04 jimoles 0s04 or 0.08 jimoles glutaraldehyde HS CH 2 CH COO NH, Fig. 4. Structures of zinc-cysteine complex and zinc cysteinate, the two possible chemical forms of the beagle tapetal rod material WAVELENGTH mp 600 Fig. 3. Spectra of cysteine obtained after reaction with acid ninhydrin showing the effect of added osmium tetroxide or glutaraldehyde. dehyde in S0rensen's phosphate buffer for 2 to 24 hr and was then postfixed in 1% osmium tetroxide and processed for EM. 7 ' 8 Another piece of tapetum was directly immersed in 1% osmium tetroxide or a mixture of equal volumes of 1% osmium tetroxide and 4% glutaraldehyde without going through fixation with glutaraldehyde alone. 4 8 ' Again, it was processed for EM in the same way. 7 Tissue preparation for zinc studies Histochemical localization of zinc. Three normal beagles and two beagles with the tapetum abnormality were perfused with 4% glutaraldehyde in S0rensen's buffer saturated with H 2 S for fixation of zinc as zinc sulfide and for ultrastructural detections as silver particles (Timm's sulfide-silver method). 9 " 12 Samples were retained for zinc measurement immediately after the perfusion, prior to processing. Other heavy metals can also be fixed by this procedure, but the content of zinc in the dog tapetum lucidum is so great that virtually all silver particles may be attributed to zinc. 13 ' H In addition, no other heavy metals could be detected in dog tapetum lucidum by atomic absorption spectrometry. 4 This method has been used successfully for determining the location of zinc in the hippocampus 12 ' 15 ~ 18 and in the feline tapetum. 13 ' 14 Retina and tapetum were prepared for examination by light microscopy and EM as described previously. 7 Measurement of zinc. The zinc content in the tapetum after fixation with the methods described above (see Table I for detailed list of samples) was measured with an atomic absorption spectrophotometer (Model ; Hitachi) by the calibration curve method. 19 Tissue preparation for measurement of cysteine and taurine. The eyes removed for biochemical measurements on fresh tissues were immediately hemisected and the vitreous was removed and discarded. The retina was carefully floated off intact in a dish of 0.9% saline, and the remaining sclera, with choroid and tapetum still attached, was blotted dry. The tapetal region was cut out with a razor blade, and for comparison, a similar-sized piece of nontapetal choroid was removed. At this point, the sclera was removed from both samples. The tissues were weighed, extracted by homogenizing with 10% trichloroacetic acid, and centrifuged at 15,000 X g for 30 min, and the clear supernatant fluid was removed for analysis. Total cysteine was determined spectrophotometrically by the acid ninhydrin method of Gaitonde, 20 which is highly specific for cysteine. The identity of the compound present in such large amounts was confirmed as cysteine by amino acid analysis with a Beckman 119 CL automatic amino acid analyzer. It was eluted at the same time and had the same high 440 to 570 m/x ratio as authentic cysteine. The concentration of taurine was measured with an automatic amino acid analyzer (Beckman 120 C) as previously described. 21 Results Normal tapetum effect of fixatives on structure. The ultrastructural appearance of the tapetum lucidum from normal beagles

5 Volume 23 Number 6 Chemical changes in tapetum of beagles 737 Fig. 5. Ultrastriictural localization of zinc in the beagle tapetum by the Timm's sulfide-silver method. Cross sections of center of tapetum from normal beagle (a and c) and beagle with inherited tapetum abnormality (b and d). These tissues were fixed by H 2 S-saturated glutaraldehyde and processed as described in the text. This procedure shows the tapetal rods of the normal beagle as regular arrays of electron-dense profiles (a). Further examination at higher magnification (c) shows a substructure within these rods, consisting of two concentric rings of zinc. These exact structures are absent from the tapetal cells of the abnormal beagles, although a few electron-dense profiles with a similar diameter were observed. Whether such profiles are altered tapetal rods or other structures is unknown, (a and b, bars = 1 /xni; c and d, bars = 0.1 fim.) depended considerably on the exact method offixationused. When the tissue wasfixedby direct immersion in osmium tetroxide in buffer, the tapetal rods appeared as electrondense profiles surrounded by an outer membrane (Fig. 1). A small number of the rods appeared to be "empty," although the surrounding membrane was still clearly visible. Some of the electron-dense profiles had a small clearing in the center. This picture

6 738 Wen et al. Invest. Ophthalmol. Vis. Set. December 1982 Fig. 6. Appearance of tapetum after H 2 S fixation, a, Tapetum from affected beagle in which there is no difference from the usual glutaraldehyde fixation or from the fresh tissue, b, Tapetum from normal beagle, which now appears steely-blue, compared with very pale green after the usual glutaraldehyde perfusion or bright green in the fresh tissue. closely resembled that previously reported by Hebel 4 for the dog tapetum fixed directly with osmium tetroxide. He described this central small clearing as a microtubule running die length of the tapetal rod. The dimensions of these structures are similar to those of microtubules, but their identity remains to be proved. When the tissue was fixed by intracardiac glutaraldehyde perfusion or by immersion in glutaraldehyde followed by postfixation in osmium tetroxide, the tapetum lucidum appeared to consist of cells filled with regular arrays of tubular profiles of 150 to 180 nm (Fig. 2). The wall of the tube appeared to be the membrane that originally surrounded each tapetal rod. This picture is similar to that reported by Hebel 4 after glutaraldehyde fixation of dog tapetum. The different ultrastructural appearance of the dog tapetum accompanying the different methods of fixation (in contrast with the results obtained for the cat, in which the ultrastructural appearance is the same after both methods of fixation) may be the result of the different compositions of the tapetal rods in the two species; in the dog these consist of a zinc-cysteine complex or zinc cysteinate, 22 whereas in the cat they consist of riboflavin and a zinc-protein. 23 ' 24 Mechanisms of action of fixatives. The results of our more detailed studies on the fixation process (Table I, Figs. 3 and 4) supported the conclusion that glutaraldehyde reacted with amino groups and sulfhydryl groups, 23 both of which are present in cysteine. Measurement of free cysteine in the presence of increasing amounts of glutaraldehyde confirmed that such a chemical reaction took place (Fig. 3) and that one molecule of glutaraldehyde reacted with one molecule of cysteine. The acid ninhydrin method used for cysteine determination depends on the presence of amino and carboxyl groups, and the reduced amount of reaction product as measured at 560 mju. indicated that the amino group of cysteine had reacted with glutaraldehyde. The stoichiometry of the reaction confirmed that both the amino group and the

7 Volume 23 Number 6 Chemical changes in tapetum of beagles 739 sulfhydryl group reacted with glutaraldehyde. If the tissue is fixed directly with osmium tetroxide, the free sulfhydryl groups of the cysteine moiety react with the osmium tetroxide, resulting in the electron-dense appearance of the tapetal rods in the electron microscope. The stoichiometry of the reaction of osmium tetroxide with cysteine (one molecule of osmium tetroxide reacted with two molecules of cysteine [Fig. 3]) confirmed that this material reacted only with the sulfhydryl group of cysteine. This explanation was supported by the results obtained after perfusion with glutaraldehyde saturated with H 2 S forfixationof zinc as zinc sulfide (Fig. 5). Under these conditions, the amino and sulfhydryl groups of cysteine react with glutaraldehyde and leave no group available for later reaction with osmium tetroxide. The zinc sulfide produced during this process was later converted to silver particles, which were seen as electrondense material in the electron microscope. Postfixation with osmium tetroxide did not alter the electron microscopic appearance of the tapetal rods in this method. It was of note that the tapetal rods fixed in this manner were not uniformly electron dense but rather consisted of two concentric rings, seen more easily in the high magnification electron micrographs (Fig. 5, c). In the center of each rod was a clear area of 16 to 20 nm diameter, which corresponded to the previously mentioned microtubule-like structure. Another aspect of note is that when H 2 S was included in the glutaraldehyde, the color of the tapetum was changed to a steely-blue (Fig. 6, b). Measurement of zinc content in the variously fixed tissue (Table I) indicated that a substantial amount of zinc remained in the tissue after all of the fixation methods used. Only during immersion fixation directly in osmium tetroxide was any reduced zinc content noted. This result suggests that zinc is present chemically in the dog tapetum as zinc cysteinate rather than as a zinc-cysteine complex. Abnormal tapetum. Ultrastructural examination of the tapetum from the affected beagles confirmed the observation that such Table I. Zinc in beagle tapetum Tapetum sample Normal fresh (n = 4) Affected fresh (n = 4) Normal fixed Dog 1 (perfused) G G, 24 hr in G G, 24 hr in B Dog 2 (immersed) G, 2hr G, 24 hr Dog 3 (perfused) G/H 2 S G/H 2 S, 24 hr in G G/H 2 S, 24 hr in B Dog 4 (immersed) 2% OsO 4> 24 hr in B G/1% OsO 4, 24 hr in B Zinc content (ppt) 26.2 ± ± G = glutaraldehyde; B = buffer; ppt = parts per thousand. cells were indeed present, although they were abnormally shaped 3 (Fig. 2, b). Notably, the micrographs of the tapetum shown here depicted far more irregularly shaped cells than illustrated previously, 3 and the contents of these cells were far more irregular than those shown previously. The explanation probably rests with the age of the beagles used, ours being at least 10 months old compared with 12 weeks old, since the abnormality is a progressive one. The large spaces between tapetal cells were filled with fixable material, including arrays of highly organized material resembling collagen. The material fixed with glutaraldehyde either by perfusion or by immersion and postfixation with osmium tetroxide in the usual way showed an absence of the tubular profiles that were present in the tapetum of normal beagles (Fig. 2, b and d). Instead, the cytoplasm was filled with membrane-bound vacuoles of various sizes, which had no apparent regularity in organization. There were many various-sized electron-dense profiles and myelin-like fragments between these vacuoles. Fixation by direct immersion in osmium tetroxide showed different structure from that obtained by fixation in glutaraldehyde alone (Figs. 1, d, and 2, d). Certainly no electrondense profiles such as those seen in normal beagles were present. Fixation with glutaral-

8 740 Wen et al. Invest. Ophthalmol. Vis. Sci. December 1982 Table H. Sulfuir amino acids in beagle eyes with hereditary Normal (n = 8) Affected (n = 6) Heterozygote (n = 4) * Data expressed as /xmoles/gm wet weight. Taurine 5.48 ± ± ± 0.62 Tapetum Cysteine 241 ± ± 21 tapetal abnormality* Taurine 3.69 ± ± ± 0.41 Choroid Cysteine dehyde saturated with H 2 S showed the complete absence of the regular arrays of electron-dense tapetal rods observed in normal beagles (Fig. 5, b and d). This observation was confirmed by the absence of any significant amounts of zinc detectable by atomic absorption spectrometry (Table I). The gross appearance of the tapetum of the affected beagles fixed with H 2 S did not appear different from that fixed normally or from that of freshly dissected eyes (Fig. 6, a). The absence of zinc in the tapetal rods of the affected beagles was accompanied by the complete absence of cysteine (Table II), which was normally present in very large amounts. However, the taurine concentration in the tapetum of the affected beagles was unchanged (Table II). Chemical composition of tapetum. The major chemical substances present in the normal beagle (homozygous dominant) tapetum were zinc and cysteine (Tables I and II). Zinc was virtually absent from the tapetum of affected beagles (homozygous recessive), whereas it was present in the tapetum of normal beagles in large amounts (Table I). The cysteine content in the tapetum of normal and heterozygous beagles was not different (Table II). No cysteine was detected in the tapetum of the affected beagles. The taurine concentration in the tapetum of the affected beagles was not different from that in the tapetum of normal or heterozygous beagles (Table II). Discussion Our results established that the defect in the tapetum of the affected beagles was an absence of the regular arrays of tapetal rods and the absence of the zinc-cysteine complex or zinc cysteinate, which constitutes the core of such normal tapetal rods. The presence of zinc as part of the tapetal rod core structure in the dog is quite different from its location in the tapetum of the cat, where it is present outside the tapetal rods, either as part of the tapetal rod membrane, or between the membrane and the rod 25 The peripheral location in the cat tapetal rods vs. the interior location in the dog tapetal rods is in accord with the original observations of Weitzel et al., 26 which we confirmed, that the zinc content of dog tapetum is higher by an order of magnitude than that in cat tapetum. The different methods of tissue fixation used in this work provided some additional information about the ultrastructure of the tapetum. The zinc-cysteine complex or zinc cysteinate, which composes the bulk of the tapetal rod core in the dog, reacted differently with thefixativesto give different electron microscopic appearances of this tissue. To visualize zinc cysteinate, the sulfhydryl group must be free to react with osmium tetroxide. This occurs during direct immersion in osmium tetroxide (Fig. 1, a and c). Fixation with glutaraldehyde, either by perfusion or by immersion, involves chemical reaction with both the amino group and the sulfhydryl group of the cysteine moiety. Both the sulfhydryl and amino groups would be expected to react with glutaraldehyde whether the dog tapetal rods consisted of a zinc-cysteine complex or of zinc cysteinate 26 (Fig. 4). As a result, the postfixation process with osmium tetroxide is ineffective, since there are no remaining free sulfhydryl groups (Fig. 2, a and c). The result, therefore, was different from that with postfixation of other macromolecular structures. Inclusion of H 2 S in

9 Volume 23 Number 6 Chemical changes in tapetum of beasj.es 741 the glutaraldehyde perfusion caused the zinc moiety to be converted to zinc sulfide, a reducing agent that was later utilized to convert silver nitrate to metallic silver, which is visualized by EM (Fig. 5, a and c). Direct measurement of the zinc content in tapetal tissue after these various methods of fixation suggests that the bulk of the zinc remained in the tissue (Table I). Only when osmium tetroxide was used directly was there any suggestion of a lowered zinc content. The somewhat greater values obtained for fixed tissue compared with fresh tissue may be attributed, at least in part, to the loss of water that occurs during fixation, since values are based on tissue weight. Direct fixation with osmium tetroxide and fixation with H 2 S- saturated glutaraldehyde fix and visualize the zinc cysteinate within the core of the tapetal rods. Suchfixationalso reveals an interesting substructure, which suggests that zinc is not uniformly distributed within the tapetal rods but rather is present in two concentric rings, with a microtubule-like structure running through the core. The reason for such a distribution is not apparent. Perfusion with H 2 S- saturated glutaraldehyde, by altering the chemical nature of the zinc within the tapetal rod core, changes the color of the tapetum reflection. The abnormally shaped tapetal cells from beagles with the inherited abnormality were filled with membrane-bound vacuoles and other material. The concentration of taurine was not different from that in normal beagles, which on the premise that taurine was associated with such membranes, suggests that it is not the lack of membrane that results in the abnormality. Possibly, the various-sized membrane-bound vacuoles result from the membrane that would otherwise surround tapetal rods. This tapetal abnormality in beagles was different from the degeneration observed in cats fed a taurine-free diet. 2 In the latter case, the depletion of taurine from the tapetum resulted in fragmentation of the membrane surrounding the tapetal rods, which in turn led to loss of structure of the rods and ultimately to degeneration of the tapetal cells. All observations of ultras tructure and chemistry of the tapetum from beagles known to be heterozygotes for this condition were indistinguishable from those of normal beagles (Table II), supporting the original suggestion that this condition is inherited as an autosomal recessive trait. 3 The peculiar chemical structure of the tapetum of the dog, in which it has both the highest zinc content and the highest cysteine content of any mammalian tissue, has a number of implications. Any treatment that causes disruption of the chemical balance, as apparently occurs with ethambutol, 28 " 31 a drug used for treating tuberculosis and with zinc pyridinethione, 32 ' 33 an antifiingal agent used in shampoos for treating dandruff, may result in adverse reactions in the dog. Ethambutol chelates the zinc in the tapetum and actually diminishes the tapetal zinc content, resulting in ultrastructural disorganization and loss of tapetum color. These changes are reversible. Ethambutol does not react with sulfhydryl groups and may remove both the zinc and the cysteine from the tapetal rods. This drug presumably exposes other tissues to toxic actions of cysteine. 34 " 36 The dramatic effects of zinc pyridinethione, which causes degeneration of the tapetum and retina followed by blindness, are absent in beagles with the hereditary abnormality, which is to be expected, since there is no cysteine to be released. The mechanism involved in the accumulation of these massive amounts of zinc and cysteine in the dog tapetum is entirely unknown but is likely to be an intriguing one. We gratefully acknowledge the expert technical assistance of Ms. Rebecca Barbour, Ms. Carol Kashdan, Mr. S. Altomare, Mr. A. Love, Mr. R. Saperstein, and Mr. J. Westheimer during this study. We are grateful to Mr. Richard Weed for expert photography. REFERENCES 1. Pirie A: The chemistry and structure of the tapetum lucidum in animals. In Aspects of Comparative Ophthalmology, Graham-Jones O, editor., New York, 1966, Pergamon Press, pp Wen GY, Sturman JA, Wisniewski HM, Lidsky AA, Cornwell AC, and Hayes KC: Tapetum disorganization in taurine-depleted cats. INVEST OPHTHALMOL VIS SCI 18:1201, Bellhorn RW, Bellhorn MB, Swarm RL, and Impel-

10 742 Wen et al. Invest. Ophthalmol. Vis. Sci. December 1982 lizzeri CW: Hereditary tapetal abnormality in beagle. Ophthalmic Res 7:250, Hebel R: Entwicklung und Strucktur der Retina und des Tapetum lucidum des Hundes. Adv Anat Embryol Cell Biol 45:7, Aguirre G: Retinal degenerations in the dog. I. Rod dysplasia. Exp Eye Res 26:233, Kuwabara T: Species differences in the retinal pigment epithelium. In The Retinal Pigment Epithelium, Zinn and Marmor MF, editors. Cambridge, Mass., 1979, Harvard University Press, pp Wisniewski HM and Bloom BR: Experimental allergic optic neuritis (EAON) in the rabbits. J Neurol Sci 24:257, Glauert AM: Fixation, dehydration and embedding of biological specimens. In Practical Methods in Electron Microscopy. New York, 1974, Elsevier/ North-Holland, Inc. 9. Pihl E and Falkmer S: Trials to modify the sulfidesilver method for ultrastructural tissue localization of heavy metals. Acta Histochem 27:34, Timm F: Zur Histochemie der schwermetalle. Das Sulfid-Silberverfahren Dtsch Z Gericht Med 46:706, Timm F: Zur Histochemie des Ammonshorngebietes. Z Zellforsch 48:548, Ibata Y and Otsuka A: Electron microscopic demonstration of zinc in the hippocampal formation using Timm's sulfide-silver technique. J Histochem Cytochem 17:171, Kohler T: Histochemical and cytochemical demonstration of zinc cysteinate in the tapetum lucidum of the cats. Histochemistry 70:173, Sturman JA, Wen GY, Wisniewski HM, and Hayes KC: Histochemical localization of zinc in the feline tapetum: effect of taurine depletion. Histochemistry 72:341, Von Euler C: On the significance of the high zinc content in the hippocampal formation. In Physiologie de l'hippocampe, Passouant P, editor. Cell Intern du CNRS no. 107, Paris, 1962, Paris Ed du Centre Nat Rech Sci pp Nicklowitz WJ: Cytomorphological alterations of mossy fiber bautons of the hippocampus produced by 3-acetylpyridine, methoxypyridine, reserpine and iproniazid. Z Zellforsch 101:192, Dreosti IE, Manvel SJ, Buckley RA, Fraser FJ, and Record IR: The effect of late prenatal and/or early postnatal zinc deficiency on the development and some biochemical aspects of the cerebellum and hippocampus in rats. Life Sci 28:2133, Danscher G: Histochemical demonstration of heavy metals. Histochemistry 71:1, Parker MM, Humoller FL, and Mahler DJ: Determination of copper and zinc in biological material. Clin Chem 13:40, Gaitonde MK: A spectrophotometric method for the direct determination of cysteine in the presence of other naturally occurring amino acids. Biochem J 104:627, Sturman JA: Taurine pool sizes in the rat: effects of vitamin B 6 deficiency and high taurine diet. J Nutr 103:1566, Weitzel G, Buddecke E, Fretzdorff AM, Strecker FJ, and Roester U: Struktur der im Tapetum lucidum von Hund und Fuchs enthaltenen zinkverbindung. Hoppe Seylers Z Physiol Chem 299:193, Elliott JH and Futterman S: Fluorescence in the tapetum of the cat's eye. Arch Ophthalmol 70:531, Croft LR: Isolation of a zinc protein from the tapetum lucidum of the cat. Biochem J 130:303, Hayat MA: Principles and Techniques of Electron Microscopy. New York, 1981, Von Nostrand Reinhold Co., vol Weitzel G: Chemie and Physiologie biogener Zinc- Verbinduungen. Agnew Chem 68:566, Weitzel G, Strecker FJ, Roester U, Buddecke E, and Fretzdorff AM: Zinc in tapetum lucidum. Hoppe Seylers Z Physiol Chem 296:19, Vogel AW and Kaiser JA: Ethambutol-induced transient change and reconstitution (in vivo) of the tapetum lucidum color in the dog. Exp Mol Pathol 2(Suppl.):136, Kaiser JA: A one year study of the toxicity of ethambutol in dogs: results during life. Toxicol Appl Pharmacol 6:557, Capiello VP and Lay ton WM: A one year study of the toxicity of ethambutol in dogs: results of gross and histopathologic examination. Toxicol Appl Pharmacol 7:844, Figueroa R, Weiss H, Smith JC, Hackley BM, Mc- Bean LD, Swassing CR, and Holstad JD: Effect of ethambutol on the ocular zinc concentration in dogs. Am Rev Respir Dis 104:592, Snyder FH, Buehler EV, and Winek CL: Safety evaluation of zinc 2-pyridinethiol 1-oxide in a shampoo formulation. Toxicol Appl Pharmacol 7:425, Cloyd GG, Wyman M, Shadduck JA, Winrow MJ, and Johnson GR: Ocular toxicity studies with zinc pyridinethione. Toxicol Appl Pharmacol 45:771, Olney JW and Ho OL: Brain damage in infant mice following oral intake of glutamate, aspartate or cysteine. Nature 227:609, Olney JW, Ho OL, Rhee V, and Schainker B: Cysteine-induced brain damage in infant and fetal rodents. Brain Res 45:309, Pedersen OO and Karlsen RL: The toxic effect of L-cysteine on the rat retina: a morphological and biochemical study. INVEST OPHTHALMOL VIS SCI 19:886, 1980.